Slow release protein polymers

ABSTRACT

The invention features articles for delivery of a biologically active substance, methods for making such articles, and methods for treating an animal using the articles.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of and claims priority fromU.S. Ser. No. 09/772,174, filed Jan. 29, 2001; which claims benefit ofU.S. S. No. 60/178,852, filed Jan. 28, 2000, entitled “Slow ReleaseProtein Polymers,” having as inventors Steven C. Rowe, Kalvin Yim,Beadle P. Retnarajan, and Jeffrey A. Hubbell.

BACKGROUND OF THE INVENTION

[0002] The invention relates to biodegradable compositions forsustained-release drug delivery and methods for administering abiologically active substance via these compositions.

[0003] Rapid advances in the fields of genetic engineering andbiotechnology have led to the development of an increasing number ofproteins and polypeptides that are useful as pharmaceutical agents. Thedevelopment of methods for administering these new pharmaceutical agentsis thus becoming increasingly important.

[0004] Most proteins have relatively short half-lives, requiringfrequent administration to achieve efficacious blood levels. To increasepatient convenience and to improve efficacy and safety by keeping bloodlevels within the therapeutic range, smoothly releasing injectable depotformulations of protein drugs are highly desirable.

[0005] Recent polymer developments have improved the ability to deliverproteins and peptides by allowing for slower and steadier release of themolecule in the patient's system. However, in many cases, the activeform of the protein is difficult to formulate in biodegradable polymers.Synthetic materials, such as biodegradable hydrogels, have also beendeveloped for use in delivering proteins. Despite the advances providedby the available polymers and hydrogels, the delivery of protein to thesystemic and local circulation is still relatively rapid, in some casestoo rapid to allow this route of administration to be used.

SUMMARY OF THE INVENTION

[0006] The present invention features articles for delivery of abiologically active substance (hereafter “BAS”), and methods for makingsuch articles. The articles of the invention improve the bioavailabilityof the BAS by formulating the BAS in an insoluble form. The inventionalso features methods of treating an animal using the articles fordelivery of a BAS.

[0007] Accordingly, in a first aspect the invention features abiocompatible therapeutic article for delivery of a BAS, comprising amacromer, a molecule or mixture of molecules which preferentiallyexcludes proteins, and the BAS, wherein the BAS is in an insolubleformat upon completion of the formulation of the article comprising themacromer, molecule, or mixture of molecules which preferentiallyexcludes proteins, and BAS.

[0008] In a preferred embodiment of the first aspect of the invention,the biocompatible therapeutic article has at least one of the followingproperties: the BAS is less than 15% aggregated; the article contains atleast 10% macromer and at least 5% BAS, as measured by dry weight; thetime at which 5% of the releasable BAS is released from the article isgreater than {fraction (1/16)} of t₅₀; or the t₅₀ is greater than orequal to ⅝ of t₈₀. More preferably the biocompatible therapeutic articlehas at least two of the above properties. Most preferably, thebiocompatible therapeutic article has all of the above properties.

[0009] In another embodiment of the first aspect of the invention, themolecule which preferentially excludes proteins is a macromer,poly(ethylene glycol), hyaluronic acid, or poly(vinylpyrrolidone). Inyet another embodiment, the macromer is a hydrogel. In still anotherembodiment, the solubility of a protein in the article comprising themacromer, molecule that preferentially excludes proteins, and BAS isless than 5-10 mg/ml, and more preferably is less than 1 mg/ml.

[0010] In another embodiment of the first aspect of the invention, themixture of molecules comprises a positively charged ion-carryingreagent, for example, triethanolamine or Tris, when the pH is such thatthe protein is negatively charged. In still another embodiment, themixture of molecules comprises a negatively charged ion-carryingreagent, such as sodium dodecyl sulfate, when the pH is such that theprotein is positively charged. In yet another embodiment, the mixture ofmolecules comprises a surfactant, for example, Tween 20, Tween 80, orpoloxamer F68. In a second aspect, the invention features a method formaking a therapeutic article for delivery of a BAS, involving (a)combining the BAS with a molecule or mixture of molecules whichpreferentially excludes proteins; (b) combining the mixture formed instep (a) with a macromer, wherein the BAS is in an insoluble form andremains insoluble upon combining with the molecule or mixture ofmolecules which preferentially excludes proteins and the macromer; (c)forming a mixture of the combination formed in step (b); and (d)polymerizing the mixture to form an article.

[0011] In one embodiment of the second aspect of the invention, steps(a) and (b) are combined into a single combination step.

[0012] In a preferred embodiment of the second aspect of the invention,the biocompatible therapeutic article has at least one of the followingproperties: the BAS is less than 15% aggregated; the article contains atleast 10% macromer and at least 5% BAS, as measured by dry weight; thetime at which 5% of the releasable BAS is released from the article isgreater than {fraction (1/16)} of t₅₀; or the t₅₀ is greater than orequal to ⅝ of t₈₀. More preferably the biocompatible therapeutic articlehas at least two of the above properties. Most preferably, thebiocompatible therapeutic article has all of the above properties.

[0013] In another embodiment of the second aspect of the invention, themolecule which preferentially excludes proteins is a macromer,poly(ethylene glycol), hyaluronic acid, or poly(vinylpyrrolidone). Inyet another embodiment, the macromer is a hydrogel. In yet anotherembodiment, the macromer is a hydrogel. In still another embodiment, thesolubility of a protein in the article comprising the macromer, moleculethat preferentially excludes proteins, and BAS is less than 5-10 mg/ml,and more preferably is less than 1 mg/ml.

[0014] In another embodiment of the second aspect of the invention, themixture of molecules comprises a positively charged ion-carryingreagent, for example, triethanolamine, when the pH is such that theprotein is negatively charged. In still another embodiment, the mixtureof molecules comprises a negatively charged ion-carrying reagent, suchas sodium dodecyl sulfate, when the pH is such that the protein ispositively charged. In yet another embodiment, the mixture comprises asurfactant, for example, Tween 20, Tween 80, or poloxamer F68.

[0015] In a third aspect the invention features a method of treating ananimal, involving administering the biocompatible therapeutic article ofthe first aspect of the invention to a mammal. Preferably the mammal isa rodent, and most preferably the mammal is a human.

[0016] In yet other preferred embodiments, the articles are administeredto the lung of the mammal, or are administered intravenously,subcutaneously, intramuscularly, orally, or nasally.

[0017] In a preferred embodiment of any of the above aspects of theinvention, the macromer comprises: (a) a region forming a central core;(b) at least two degradable regions attached to the core; and (c) atleast two polymerizable end groups, where the polymerizable end groupsare attached to the degradable regions. In preferred embodiments, theregion forming a central core is a water soluble region. The watersoluble region may be poly(ethylene glycol), poly(ethylene oxide),poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline),poly(ethylene oxide)-co-poly(propylene oxide) block copolymers,polysaccharides, carbohydrates, proteins, and combinations thereof. Thedegradable region is selected from the group consisting ofpoly(α-hydroxy acids), poly(lactones), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(orthocarbonates), andpoly(phosphoesters). Preferably, the poly(α-hydroxy acid) ispoly(glycolic acid), poly(DL-lactic acid), or poly(L-lactic acid), andthe poly(lactone) is poly(β-caprolactone), poly(δ-valerolactone), orpoly(γ-butyrolactone). In another preferred embodiment, the degradableregion comprises poly(caprolactone). In yet another embodiment, thepolymerizable end groups contain a carbon-carbon double bond capable ofpolymerizing the macromer.

[0018] In other embodiments of the above aspects of the invention, themacromer includes: (a) a water soluble region comprising a three-armedpoly(ethylene glycol) with a molecular weight of 3,000 to 6,000 daltons;(b) lactate groups attached to the region in (a); and (c) acrylategroups capping the region in (b). The macromer may alternativelyinclude: (a) a water soluble region comprising poly(ethylene glycol)with a molecular weight of either 2,000 or 3,400 daltons; (b) lactategroups on either side of the region in (a); and (c) acrylate groupscapping either side of the region in (b). In another alternative, themacromer may include (a) a water soluble region comprising poly(ethyleneglycol) with a molecular weight of 3,400 daltons; (b) caprolactonegroups on either side of region in (a); and (c) acrylate groups cappingeither side of the region in (b).

[0019] In still other embodiments of any of the above aspects of theinvention, the article includes at least 5%, more preferably 10%, andmost preferably 20-30% active substance by dry weight. In still anotherembodiment, the article is biodegradable.

[0020] In a more preferred embodiment of any of the above aspects of theinvention, the macromer includes a water soluble region consisting of athree-armed PEG with a molecular weight of 4,200 to 5,400 daltons;lactate groups one end of each arm of the PEG; and acrylate groupscapping the lactate groups.

[0021] In another more preferred embodiment of the above aspects of theinvention, the macromer is made of a triad ABA block copolymer ofacrylate-poly(lactic acid)-PEG-acrylate-poly(lactic acid)-acrylate. ThePEG has a MW of 3,400 daltons; the poly(lactic acids) on both sides hadan average of about five lactate units per side; and the macromer istherefore referred to herein as “3.4kL5.” In another more preferredembodiment, a lower molecular weight PEG, such as MW 2,000 daltons PEGis used in place of the MW 3,400 PEG, and the resulting macromer isabbreviated as “2kL5.”

[0022] In yet another more preferred embodiment of the above aspects ofthen invention, the macromer is an acrylate-PCL-PEG-PCL-acrylatemacromer. The PEG has a MW of 3,400 daltons and has polycaprolactone onboth sides, with an average of about 6 caproyl units per side. Thismacromer is referred to herein as “3.4kC6.”

[0023] In other preferred embodiments, the BAS is a protein or peptide.More preferably the protein is chosen from a group consisting ofhormones, antibodies, differentiation factors, angiogenic factors,enzymes, cytokines, chemokines, interferons, colony-stimulating factors,and growth factors. Most preferably, the protein is a hormone, such ashuman growth hormone, or a peptide, such as LHRH.

[0024] In still other embodiments of the second and third aspects of theinvention, the therapeutic articles release at least 80% of the BAS at atime 1¼ times greater than t₅₀. At least 80% of the therapeutic articlesmay have a particle size of less than about 80 microns. The watersoluble region may consist essentially of PEG having a molecular weightof about 500 to 20,000 daltons, and more preferably, between 1,000 and10,000 daltons. The degradable region may comprise a blend of at leasttwo different polymers. In addition, the macromer may be non-degradable.

[0025] In still other embodiments of the second and third aspects of theinvention, the therapeutic article is capable of releasing the BAS forat for a period of time at least 2 times greater than t₅₀. The articleis also capable of delivering a therapeutic dose of the BAS for at for aperiod of time at least 1¼ times greater than t₅₀.

[0026] By “macromer” is meant a polymer with three components: (1) abiocompatible, water soluble region; (2) a biodegradable/hydrolyzableregion, and (3) at least two polymerizable regions.

[0027] By “biologically active substance” or “BAS” is meant a compound,be it naturally-occurring or artificially-derived, that is incorporatedinto an article and which may be released and delivered to a site.Biologically active substances may include, for example, peptides,polypeptide, proteins, synthetic organic molecules, naturally occurringorganic molecules, nucleic acid molecules, and components thereof.

[0028] By “a molecule or mixture of molecules that preferentiallyexcludes proteins” is meant a molecule or mixture of molecules, be itnaturally-occurring or artificially-derived, that, when added to asolution, confers a lower level of solubility of the protein orpolypeptide in said solution. Preferably, protein solubility will bedecreased 50-fold; more preferably, 100-fold and most preferably about200-fold. Preferably the solubility of a protein in a solution thatincludes said molecule or mixture of molecules that preferentiallyexcludes proteins is less than 5-10 mg/ml, and more preferably is lessthan 1 mg/ml.

[0029] By “substantially pure polypeptide” or “protein” s meant apolypeptide or protein that has been separated from the components thatnaturally accompany it. The terms polypeptide and protein may be usedinterchangeably. Typically, the polypeptide is substantially pure whenit is at least 60%, by weight, free from the proteins andnaturally-occurring organic molecules with which it is naturallyassociated. A substantially pure polypeptide may be obtained, forexample, by extraction from a natural source (e.g., a cell expressingthe desired polypeptide), by expression of a recombinant nucleic acidencoding a desired polypeptide, or by chemically synthesizing thepolypeptide. Purity can be assayed by any appropriate method, e.g., bycolumn chromatography, polyacrylamide gel electrophoresis, agarose gelelectrophoresis, optical density, or HPLC analysis.

[0030] A protein is substantially free of naturally associatedcomponents when it is separated from those contaminants which accompanyit in its natural state.

[0031] Thus, a protein which is chemically synthesized or produced in acellular system different from the cell from which it naturallyoriginates will be substantially free from its naturally associatedcomponents. Accordingly, substantially pure polypeptides include thosederived from eukaryotic organisms but synthesized in E. Coli or otherprokaryotes.

[0032] By “purified nucleic acid” is meant a nucleic acid that is freeof the genes which, in the naturally-occurring genome of the organismfrom which the nucleic acid of the invention is derived, flank the gene.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by PCR or restriction endonuclease digestion)independent of other sequences. It also includes recombinant DNA whichis part of a hybrid gene encoding additional polypeptide sequence.

[0033] By “biocompatible” is meant that any compound or substance whichis administered to a subject, cell, or tissue is used to treat, replace,or augment a function of the subject, cell or tissue, and is not harmfulto said function.

[0034] By “insoluble” is meant that the solubility of a compound is lessthan 1 g/100 ml in a solution. The solution may be an aqueous solution,an organic solvent, such as dimethylsulfoxide, or a mixture of aqueousand organic solvents. As used herein, a BAS is in an insoluble formatupon completion of the formulation for a therapeutic article fordelivery of the BAS. The BAS remains in an insoluble format upondelivery of the therapeutic article to a patient, and is then slowlyreleased at a controlled rate for localized or systemic delivery to thepatient. As used herein, by “aggregated” is meant that a BAS isreleasable as individual molecules. The percent of a BAS in an articlewhich is aggregated can be determined, for example, by SEC-HPLC.

[0035] By “therapeutic dose,” when referring to a BAS, is meant a plasmalevel between the minimum effective level and the toxic level.

[0036] By a “mixture” is meant a composition in which all of thecompounds contained in the composition are evenly distributed.

[0037] As used herein, by “pore size” is meant the dimensions of a spacein the intact polymer through which a macromer, component of a macromer,or a BAS potentially can pass. Pore sizes which are utilized as part ofthe invention are those smaller than the BAS as it is present in theparticular embodiment (e.g., a protein molecule, or aggregate thereof).

[0038] As used herein, by “period of release” is meant the length oftime it takes for a specified percent of the BAS to be released from anarticle. The period of release may be assessed, for example, bymeasuring the time it takes for 50% or 80% of the BAS to be releasedfrom the article.

[0039] By “low burst effect” is meant that the amount of BAS releasedfrom an article is released relatively steadily over time, rather thanat an initial fast rate, followed by a slower rate. For example, a BAShas a low burst effect (e.g., less than or equal to 20% burst) uponrelease from an article when the period of release for 5% of thereleasable BAS is greater than {fraction (1/16)} of t₅₀, or when the t₅₀is greater than or equal to ⅝ of t₈₀. In contrast to a low burstarticle, a high burst article (e.g., one which rapidly releases 30% ofthe BAS) might release 5% of its releasable BAS in less than {fraction(1/18)} of _(t50) and have a t₅₀ equal to {fraction (1/2)} of t₈₀.

[0040] A specific example of a low burst product of the presentinvention is one in which less than 20% of the BAS comes out in thefirst day for a product designed to release a BAS for 10 days.

[0041] By “t₅₀” is meant the time at which 50% of the original load ofBAS has been released. As used herein, preferably 5% of the releasableBAS is released at a time which is greater than {fraction (1/16)} oft₅₀, or the t₅₀ is greater than or equal to ⅝ of the t₈₀.

[0042] By “t₈₀” is meant the time at which 80% of the original load ofBAS has been released. As used herein, preferably 5% of the releasableBAS is released at a time which is greater than {fraction (1/16)} oft₅₀, or the t₅₀ is greater than or equal to ⅝ of the t₈₀.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 is the release profile of microspheres made withprecipitated hGH.

[0044]FIG. 2 is the release profile of hGH from 2kL5 microspheres.

[0045]FIG. 3 is a graph depicting the release of spray-dried bSA from4.2kL3-A3 microspheres.

[0046]FIG. 4 is a graph of the effects of biologically active particlesize on the release of bovine serum albumen (bSA) from 30% 3.4kL5.

[0047]FIG. 5 is a graph of the effect of biologically active particlesize and protein loading on the release of fine-ground bSA (10% loaded)and various crystalline particles (1-10% loaded) from 3.4kL5.

[0048]FIG. 6 is a graph of the effects of biologically active particlesize on the release of human growth hormone (hGH) from 30% 3.4kL5.

[0049]FIG. 7 is a graph of the effect of microsphere pore size onrelease of micronized hGH from articles.

DETAILED DESCRIPTION

[0050] The invention provides methods and compositions for theadministration of a biologically active substance (BAS) in an insolubleformat. The compositions of the invention improve the bioavailability ofthe BAS by formulating the BAS in an insoluble format. These methods andcompositions provide for the controlled, sustained delivery ofrelatively large quantities of these substances, with a low bursteffect.

[0051] Macromers

[0052] The macromers of the present invention have at least one regionforming a central core, at least one degradable (e.g., hydrolyzable)region, and at least one polymerizable region. The macromers may bewater-soluble or water insoluble. Preferably, the region forming acentral core is water soluble. If desired, the macromers may bepolymerized to form hydrogels, which are useful for deliveringincorporated substances at a controlled rate. Methods to formulatemacromers and shape them into articles are described, for example in WO99/03454, hereby incorporated by reference. An important aspect of themacromers is that the polymerizable regions are separated by at leastone degradable region. This separation facilitates uniform degradationin vivo.

[0053] The ratio between the central core region and the hydrolyzableregion of the macromer determines many of the general properties of themacromer. For example, the water solubility of the macromers can becontrolled by varying the percentage of the macromer that consists ofhydrophobic degradable groups.

[0054] There are several variations of the macromers of the presentinvention. For example, the polymerizable regions can be attacheddirectly to the degradable regions; alternatively, they can be attachedindirectly via water-soluble, nondegradable regions, with thepolymerizable regions separated by a degradable region. For example, ifthe macromer contains a single water-soluble region coupled to adegradable region, one polymerizable region can be attached to thewater-soluble region, and the other to the degradable region.

[0055] In another embodiment, a water-soluble region forms the centralcore of the macromer and has at least two degradable regions attached toit. At least two polymerizable regions are attached to the degradableregions so that, upon degradation, the polymerizable regions,particularly in the polymerized gel form, are separated. Alternatively,if the central core of the macromer is formed by a degradable region, atleast two water soluble regions can be attached to the core, andpolymerizable regions are attached to each water soluble region.

[0056] In still another embodiment, the macromer has a water-solublebackbone region, with a degradable region attached to the macromerbackbone. At least two polymerizable regions are attached to thedegradable regions, such that they are separated upon degradation,resulting in gel product dissolution. In a further embodiment, themacromer backbone region is formed of a degradable backbone regionhaving water-soluble regions as branches or grafts attached to thedegradable backbone. Two or more polymerizable regions are attached tothe water soluble branches or grafts.

[0057] In another variation, the macromer backbone may have multiplearms; e.g., it may be star-shaped or comb-shaped. The backbone mayinclude a water-soluble region, a biodegradable region, or awater-soluble, biodegradable region. The polymerizable regions areattached to this backbone. Again, the polymerizable regions must beseparated at some point by a degradable region.

[0058] Throughout the specification, the following abbreviations aresometimes used to describe the specific macromers of the invention. Inthree particular examples, a macromer having a water soluble regionconsisting of PEG with a molecular weight of 4,000 daltons, with 5lactate groups on either side of this region, capped on either side withacrylate groups, is referred to as “4kL5.” Similarly, a macromer havinga water soluble region consisting of PEG with a molecular weight of3,400 daltons, with 6 caprolactone groups on either side of this region,capped on either side with acrylate groups, is referred to as “3.4kC6.”Likewise, a macromer having a water soluble region consisting of PEGhaving a molecular weight of 5,400 daltons and 3 arms, each armcontaining 3 lactate groups, extending from this region, capped oneither side with acrylate groups, is referred to as “4.2kL3-A3.”

[0059] Water-Soluble Region

[0060] In preferred embodiments, the central core is a water solubleregion. This water soluble region of the macromer may includepoly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, polysaccharides,carbohydrates, or proteins, or combinations thereof.

[0061] The macromer preferably comprises a water soluble core regioncomprising PEG, as PEG has high hydrophilicity and water solubility, aswell as good biocompatibility. The PEG region preferably has a molecularweight of about 400 to about 40,000 daltons, and more preferably has amolecular weight of about 1,000 to about 30,000 daltons, about 1,000 toabout 20,000 daltons, or about 2,000 to about 10,000 daltons.

[0062] Degradable Region

[0063] The degradable region of the macromer may contain, for example,poly(α-hydroxy acids), poly(lactones), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(orthocarbonates) orpoly(phosphoesters), or blends or copolymers of these polymers.

[0064] Exemplary poly(α-hydroxy acids) include poly(glycolic acid),poly(DL-lactic acid), and poly(L-lactic acid). Exemplary poly(lactones)include poly(ε-caprolactone), poly(δ-valerolactone),poly(γ-butyrolactone), poly(1,5-dioxepan-2-one), and poly(trimethylenecarbonate).

[0065] The degradable region may comprise a blend of at least twodifferent polymers. Examples of copolymers include a copolymer ofcaprolactone and glycolic acid; and a copolymer of caprolactone andlactic acid.

[0066] Polymerizable Region

[0067] The polymerizable regions of the macromer preferably containcarbon-carbon double bonds capable of polymerizing the macromers. Thechoice of an appropriate polymerizable group permits rapidpolymerization and gelation. Polymerizable regions containing acrylatesare preferred because they can be polymerized using several initiatingsystems, as discussed below. Examples of acrylates include acrylate,methacrylate, and methyl methacrylate.

[0068] Polymerization of Macromers

[0069] If desired, the macromers of the present invention may bepolymerized using polymerization initiators under the influence of longwavelength ultraviolet light, visible light, thermal energy, or a redoxsystem. The polymerization can be conducted at room temperature or atlower temperatures, for example, temperatures less than 20° C. Duringpolymerization, substances such as proteins are physically incorporatedinto the resulting polymer network of the hydrogel.

[0070] Polymerization of the macromers may be initiated in situ by lighthaving a wavelength of 320 nm or longer. When the polymerizable regioncontains acrylate groups, the initiator may be any of a number ofsuitable dyes, such as xanthine dyes, acridine dyes, thiazine dyes,phenazine dyes, camphorquinone dyes, acetophenone dyes, or eosin dyeswith triethanolamine, 2,2-dimethyl-2-phenyl acetophenone, and2-methoxy-2-phenyl acetophenone.

[0071] The polymerization may also take place in the absence of light.For example, the polymerization can be initiated with a redox system,using techniques known to those of skill in the art. In some cases it isadvantageous to polymerize macromers using the redox system of theinvention, as radical initiator production occurs at reasonable ratesover a wide range of temperatures.

[0072] Initiators that can be used in the redox system include, withoutlimitation, peroxides such as acetyl, benzoyl, cumyl and t-butyl;hydroperoxides such as t-butyl and cumyl, peresters such as t-butylperbenzoate; acyl alkylsulfonyl peroxides, dialkyl peroxydicarbonates,diperoxyketals, ketone peroxide, azo compounds such as2,2′-azo(bis)isobutyronitrile (AIBN), disulfides, and tetrazenes.

[0073] Shaping of Articles

[0074] The articles of the present invention may be formed in any shapedesired. For example, the articles may be shaped to fit into a specificbody cavity. They may also be formed into thin, flat disks or particles,such as microspheres. Alternatively, the articles may be shaped, thenprocessed into the desired shape before use, or ground into fineparticles. The desired shape of the article will depend on the specificapplication.

[0075] Macromer particles may be prepared using techniques known in theart, including single and double emulsion solvent evaporation, spraydrying, and solvent extraction. As used herein, the term “particles”includes, but is not limited to, microspheres. In a microsphere, a BASis dispersed throughout the particle. The particles may have a smooth orirregular surface, and may be solid or porous. Methods for makingmicrospheres are described in the literature, for example, in U.S. Pat.No. 4,272,398, Mathiowitz and Langer (J. Controlled Release 5:13-22(1987)); Mathiowitz et al. (Reactive Polymers 6:275-283 (1987));Mathiowitz et al. (J. Appl. Polymer Sci. 35:755-774 (1988)); Mathiowitzet al. (Scanning Microscopy 4:329-340 (1990)); Mathiowitz et al. (J.Appl. Polymer Sci., 45:125-134 (1992)); and Benita et al. (J. Pharm.Sci. 73:1721-1724 (1984)), hereby incorporated by reference. In onepreferred embodiment of the present invention, the microspheres areformed into hydrogel droplets.

[0076] In solvent evaporation, described, for example, in Mathiowitz, etal., (1990), Benita et al. (1984), and U.S. Pat. No. 4,272,398, apolymer is dissolved in a volatile organic solvent, such as methylenechloride. An agent to be incorporated, either in soluble form ordispersed as fine particles, is optionally added to the polymersolution, and the mixture is suspended in an aqueous phase that containsa surface active agent such as poly(vinyl alcohol). The resultingemulsion is stirred until most of the organic solvent evaporates,leaving solid microspheres, which may be washed with water and driedovernight in a lyophilizer.

[0077] In solvent removal, as described, for example, by Park et al. (J.Controlled Release 55:181-191 (1998)), a therapeutic or diagnostic agentis dispersed or dissolved in a solution of a selected polymer in avolatile organic solvent such as methylene chloride. The mixture canthen be suspended in oil, such as silicon oil, by stirring, to form anemulsion. As the solvent diffuses into the oil phase, the emulsiondroplets harden into solid polymer microspheres.

[0078] Processes for preparing ultrafine particles of biologicalmolecules by atomizing liquid solutions of the macromolecules, dryingthe droplets formed in the atomization step, and collecting theparticles are described in PCT WO 97/41833, hereby incorporated byreference.

[0079] Spray drying is implemented by passing a homogenous mixture of aBAS, such as a therapeutic agent, and the polymerizable macromer used toform a hydrogel through a nozzle, spinning disk, or equivalent device toatomize the mixture to form fine droplets. The substance and thepolymerizable macromer may be provided in a solution or suspension, suchas an aqueous solution. The fine droplets are exposed to light to causepolymerization of the macromer and formation of the hydrogel dropletsincorporating the substance. Hydrogels may be formed according to themethods described in U.S. Pat. No. 5,410,016, hereby incorporated byreference, or other techniques known in the art of polymer chemistry.

[0080] In another embodiment, hydrogel particles are prepared by awater-in-oil emulsion process, wherein the polymerizable macromers andthe substance to be incorporated are suspended in a water-in-oilemulsion and exposed to light to polymerize the macromers to formhydrogel particles incorporating the substance, such as a BAS.Typically, polymerization may be conducted at room temperature.

[0081] The microspheres prepared using the techniques described aboveare freeze dried, so they have a long shelf life (withoutbiodegradation) and the BAS remains biologically active. Prior to usefor injectable formulations, the microspheres are reconstituted in asuitable solution, such as saline or other liquids. For pulmonarydelivery, either freeze dried or reconstituted particles may be used.

[0082] Properties of the Macromers

[0083] The articles of the present invention are biodegradable.Biodegradation occurs at the linkages within the extension oligomers andresults in fragments which are non-toxic and easily removed from thebody and/or are normal, safe chemical intermediates in the body. Thesematerials are particularly useful for the delivery of hydrophilicmaterials, since the water soluble regions of the polymer allow water toaccess the materials trapped within the polymer.

[0084] Use of the Macromers

[0085] Macromers can be shaped into articles, for example, microspheres,and these articles are capable of degrading under in vivo conditions atrates which permit the controlled release of incorporated substances.Release of such a substance may occur by diffusion of the substance fromthe polymer prior to degradation and/or by diffusion of the materialfrom the polymer as it degrades. Degradation of the polymer facilitateseventual controlled release of free macromolecules in vivo by gradualhydrolysis of the terminal ester linkages. The burst effects that aresometimes associated with other release systems are thus avoided in arange of formulations.

[0086] The rate of release of a BAS depends on many factors, forexample, the composition of the water soluble region, the degree ofpolymerization of the macromer. The rate of release of a BAS alsodepends on the rate of degradation of the degradable region of themacromer. For example, glycolic esters lead to very rapid degradation,lactic esters to somewhat slower degradation, and caprolactic esters tovery slow degradation. When the degradable region consists ofpolyglycolic acid, the release period is less than one week. When thedegradable region consists of poly(lactic acid), the release period isabout one week. When the degradable region consists of a copolymer ofcaprolactone and lactic acid or a copolymer of trimethylene carbonateand lactic acid, the release period is two to four weeks. When thedegradable region consists of poly(trimethylene carbonate) or acopolymer of caprolactone and trimethylene carbonate, the release periodis about three to eight weeks. When the degradable region consists ofpoly(trimethylene carbonate) or poly(caprolactone), the release periodis longer than about five weeks.

[0087] The precise rate of release of a BAS from an article can befurther modified by altering the ratio of hydrophilic and hydrophobiccomponents of the article. For example, a very soluble macromer willyield, after polymerization, a hydrophilic gel; hydrophilic hydrogelshave been shown to degrade more rapidly than hydrophobic ones. A blendof a hydrophilic macromer (e.g., 4kL5) with a hydrophobic waterinsoluble macromer (3.4kC6) is used to form a polymerized hydrogel. Thishydrogel will have a release rate that is in between the release rate ofa hydrogel containing only lactic acid and a hydrogel containing onlycaprolactone. A macromer in which the degradable region is a copolymerof caprolactone and lactic acid will also have a release rate which isin between the release rate of a hydrogel containing only lactic acidand a hydrogel containing only caprolactone as the primary degradablegroup. Similarly, hydrophilicity of the active substance also affect therelease rate of the BAS, with hydrophilic active substances generallyreleased faster than hydrophobic substances.

[0088] The rate of release of a given BAS from a therapeutic articledepends on the quantity of the loaded substance, as a percent of thefinal product formulation. For example, it is generally thought in thepolymer field that while a large amount of BAS loading results in alonger period of therapeutic dose delivery, it also results in a largeburst effect. Therefore, an article which is loaded with a high amountof a BAS, and which also exhibits a low burst effect would be an optimalarticle. The articles or the present invention exhibit thesecharacteristics.

[0089] Other factors which affect the release rate of a BAS from anarticle are the aggregation and the solubility of the BAS. In order forthe articles of the present invention to have release profiles which areoptimal for delivering a BAS, the percent of the BAS which is aggregatedshould be low. The articles of the present invention contain BAS whichare preferably less than 15% aggregated. In preferred embodiments, thearticles have this characterization of low aggregation even when theyand contain at least 2.5% BAS by dry weight, more preferably at least5%, and most preferably 20 or 40% by dry weight.

[0090] As stated above, another factor which affects the rate of releaseof a BAS from an article is the solubility of the BAS in the article. Inthe field of polymer chemistry, it has generally been thought thatwater-soluble substances, such as a BAS, will yield homogenous systemswhen incorporated into the macromers of the invention. It has also beenthought that substances that do not solubilize in water within the timeit takes to form the macromers of the invention will yield heterogenoussystems. While the amount of burst in the heterogenous systems can beminimized by using a particulate suspension with small particles, it isgenerally thought that substances should be in a water soluble formatfor optimal delivery in a polymer delivery system. The articles of thepresent invention contain a BAS in an insoluble format, and thesearticles exhibit a low burst effect, an unexpected result.

[0091] Yet another factor that affects the release rate of a BAS from anarticle is the particle size of the BAS. For example, the articles ofthe present invention feature a BAS which has been ground and sieved toisolate fine particles which are smaller than approximately 75 micronsin any dimension. These particles were used to generate microspheres andthe release of the BAS from the microspheres was measured. This releaserate was compared to the release rate of the same BAS from the samemicrospheres, with the exception that the BAS was not fine-ground. Theresults of these studies indicated that a BAS which is fine-groundresults in release rates which are slower and have a low burst effect.By adjusting the factors discussed above, degradation and controlledrelease may be varied over very wide ranges. For example, release may bedesigned to occur over hours, days, or months.

[0092] The methods of the invention can produce particles that behave ashomogenous drug delivery systems. Because of the homogenous nature ofthe articles of the invention, there is no initial burst of releasedsubstance. In addition, the uniform consistency makes it possible toincorporate relatively high amounts of protein, while still minimizingthe burst effect.

[0093] The present invention also features insoluble macromers. Thesemacromers contain at least one water-soluble region, at least onedegradable (e.g., hydrolyzable) region, and at least one polymerizableregion. The degradable region contains polymers of glycolic acid, lacticacid, caprolactone, trimethylene carbonate, or blends or copolymersthereof. The degradable region must be water insoluble. For example, amacromer having a degradable region containing 15-20 lactide units canbe prepared; this macromer will provide a relatively fast release rate.A macromer with a degradable region containing 6 caprolactone units willprovide a relatively slow release rate. A macromer with a degradableregion containing a copolymer of 6 caprolactone units, 4 lactide units,and 4 glycolide units will provide a fast release rate, and a macromerwith a degradable region containing a copolymer of 3 lactide units and 7trimethylene carbonate units will provide an intermediate release rate.

[0094] The water soluble region of these macromers is preferably PEG.The water soluble region can have multiple arms; for example, it may bestar-shaped or comb-shaped, as described, for example in U.S. Pat. No.5,410,016, incorporated herein by reference. The water soluble regionpreferably has 3, 4, 6, or 8 arms and a molecular weight of 500 to20,000, preferably, 1,000 to 10,000 daltons.

[0095] Methods for Increasing Protein Precipitation

[0096] The articles of the present invention, can be made to contain aBAS in an insoluble format, by combining the BAS with a molecule, ormixture or molecules which preferentially excludes proteins, and amacromer, forming a mixture of these reagents, and polymerizing themixture. A molecule or mixture of molecules which preferentially excludeproteins can be used in the formation of the article to increase proteinprecipitation. Examples of molecules which preferentially excludeproteins include, but are not limited to, macromers, poly(ethyleneglycol), hyaluronic acid, and poly(vinylpyrrolidone). A reagent whichcarries a positive or negative ion charge may be used in the formationof the articles of the invention in order to increase the precipitationof the BAS in the mixture which is then polymerized to form the article.The optimal reagent to be used depends on the charge of the protein,which is affected by the pH of the mixture. Examples of mixtures ofmolecules which preferentially exclude proteins include, but are notlimited to, a mixture of molecules comprising a positively chargedion-carrying reagent, for example, triethanolamine or Tris (for example,when the pH is such that the protein is negatively charged); or amixture of molecules comprising a negatively charged ion-carryingreagent, such as sodium dodecyl sulfate (for example, when the pH issuch that the protein is positively charged). A mixture comprising asurfactant, for example, Tween 20, Tween 80, or poloxamer F68, may alsobe used to increase the precipitation of the protein.

[0097] High Load and Low Burst Characteristics

[0098] A therapeutic agent, for example, a BAS may be readilyincorporated in high yield into the articles described herein. Forexample, articles may be prepared containing at least 5% activesubstance by dry weight. Preferably, the articles contain at least 10,25, or 40% by dry weight.

[0099] As discussed above, the BAS of the present invention is in aninsoluble format when combined with a macromer and formed into anarticle. The combination of high load and the insoluble format of theactive substance in the article provides the article with a slow releaseprofile, with little initial burst. These results are surprising giventhe view in the field of polymers that an article containing aninsoluble active substance will have large initial burst of the activesubstance.

[0100] The BAS contained in the articles of the present invention isinsoluble. The formulation of articles containing an insoluble BAS maybe achieved, for example, by mixing the BAS with PEG, and then combiningthese reagents with the desired macromer.

[0101] The amount of BAS loaded into a microsphere may be measured bycombining it with a macromer and shaping into articles. The articles maythen be placed into an appropriate solvent, for example phosphatebuffered release media (0.01% NaN₃, 0.05 M PBS, pH 7.4) and assayed forthe amount of BAS present by means available in the art, such asspectrophotometry.

[0102] Biologically Active Substances

[0103] A BAS that can be incorporated into the articles of the inventioninclude therapeutic, diagnostic, and prophylactic agents. They can benaturally occurring compounds, synthetic organic compounds, or inorganiccompounds. Substances that can be incorporated into the articles of theinvention include proteins, polypeptides, carbohydrates, inorganicmaterials, antibiotics, antineoplastic agents, local anesthetics,antiangiogenic agents, vasoactive agents, anticoagulants,immunomodulators, cytotoxic agents, antiviral agents, antibodies,neurotransmitters, psychoactive drugs, oligonucleotides, lipids, cells,tissues, tissue or cell aggregates, and combinations thereof.

[0104] Exemplary therapeutic agents include growth hormone, for examplehuman growth hormone, calcitonin, granulocyte macrophage colonystimulating factor (GMCSF), ciliary neurotrophic factor, parathyroidhormone, and the cystic fibrosis transmembrane regulator gene. Otherspecific therapeutic agents include parathyroid hormone-relatedpolypeptide, somatostatin, testosterone, progesterone, estradiol,nicotine, fentanyl, norethisterone, clonidine, scopolomine, salicylate,salmeterol, formeterol, albeterol, and valium.

[0105] Drugs for the treatment of pneumonia may be used, includingpentamidine isethionate. Drugs for the treatment of pulmonaryconditions, such as asthma, may be used, including albuterol sulfate,β-agonists, metaproterenol sulfate, beclomethasone dipropionate,triamcinolone acetamide, budesonide acetonide, ipratropium bromide,flunisolide, cromolyn sodium, ergotamine tartrate, and protein orpolypeptide drugs such as TNF antagonists or interleukin antagonists.

[0106] Other therapeutic agents include cancer chemotherapeutic agents,such as cytokines, chemokines, lymphokines, and substantially purifiednucleic acids, and vaccines, such as attenuated influenza virus.Substantially purified nucleic acids that can be incorporated includegenomic nucleic acid sequences, cDNAs encoding proteins, expressionvectors, antisense molecules that bind to complementary nucleic acidsequences to inhibit transcription or translation, and ribozymes. Forexample, genes for the treatment of diseases such as cystic fibrosis canbe administered. Polysaccharides, such as heparin, can also beadministered.

[0107] Other therapeutic agents include tissue plasminogen activator(t-PA), superoxide dismutase, catalase luteinizing hormone releasinghormone (LHRH) antagonists, IL-11 platelet factor, IL-4 receptor,enbrel, IL-1 receptor antagonists, TNF receptor fusion proteins,megakaryocyte growth and development factor (MGDF), stemgen, anti-HER-2and anti-VEGF humanized monoclonal antibody, anti-Tac antibody, GLP-1amylin, and GLP-1 amylin analogues.

[0108] Additional therapeutic agents include atrial natriuretic factor,atrial natriuretic peptide, beta-human chorionic gonadotropin, basicfibroblast growth factor, bovine growth hormone, bone morphogeneticprotein, B cell stimulating factor-1, B cell stimulating factor-2,bovine somatotropin, carcinobreaking factor, cartilage induction factor,corticotropin releasing factor, colony stimulating factor,differentiating factor-1, endothelial cell growth factor, erythroiddifferentiation factor, elongation factor 1-alpha, epidermal growthfactor, erythropoietin, thrombopoietin, thymopoietin, fibroblast growthfactor, follicle stimulating hormone, granulocyte colony stimulatingfactor, glial fibrillary acidic protein, growth hormone releasingfactor, human alpha-1 antitrypsin, human atrial natriuretic factor,human chorionic gonadotropin, human leukemia inhibitory factor,hemopoietin-1, hepatocyte growth factor, human transforming growthfactor, human thyroid-stimulating hormone, interferon, immunoglobulin A,immunoglobulin D, immunoglobulin E, insulin-like growth factor-1,insulin-like growth factor-II, immunoglobulin G, immunoglobulin M,interleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6, kidney plasminogen activator, lectin celladhesion molecule, luteinizing hormone, leukemia inhibitor factor,monoclonal antibody, macrophage activating factor, macrophage cytotoxicfactor, macrophage colony stimulating factor, megakaryocyte colonystimulating factor, tumor necrosis factor, macrophage inhibitory factor,Mullerian inhibiting substance, megakaryocyte stimulating factor,melanocyte stimulating factor, neutrophil chemotactic factor, nervegrowth factor, novel plasminogen activator, nonsteroidalanti-inflammatory drug, osteogenic factor extract, antitumor lymphokine,prostate-specific antigen, anti-platelet activating factor, plasminogenactivator inhibitor, platelet-derived growth factor, platelet-derivedwound healing formula, plasmatic human interleukin inducing protein,tumor angiogenesis factor, tissue control factor, T cell growth factor,T cell modulatory peptide, transforming growth factor, tumor growthinhibitor, tumor inhibiting factor, tissue inhibitor ofmetalloproteinases, tumor necrosis factor, tissue plasminogen activator,thyroid stimulating hormone, urokinase-plasminogen activator, vascularendothelial growth factor, and vasoactive intestinal peptide.

[0109] A preferred BAS is a substantially purified polypeptide orprotein. Proteins are generally defined as consisting of 100 amino acidresidues or more; polypeptides are less than 100 amino acid residues.Unless otherwise stated, the term protein, as used herein, refers toboth proteins and polypeptides. The proteins may be produced, forexample, by isolation from natural sources or recombinantly. Examplesinclude insulin and other hormones, including growth hormones, such ashuman growth hormone and bovine growth hormone. Other exemplary proteinsinclude Factor VIII, Factor IX, Factor VIIa, and anti-inflammatoryagents, such as interleukins, including interleukin-4. Other exemplaryproteins include enzymes, such as DNase and proteases. Other proteinsinclude cytokines, interferons, including interferon alpha andinterferon beta, poetins, angiogenic factors, differentiation factors,colony-stimulating factors, growth factors, ceredase, gibberellins,auxins, and vitamins, and fragments thereof. Exemplary growth factorsinclude vascular endothelial growth factor (VEGF), endothelial cellgrowth factor (ECGF), basic fibroblast growth factor (bFGF), andplatelet derived growth factor (PDGF).

[0110] Proteins are stable in the hydrogels of the present invention.For example, many of the proteins are protected from dimerization oraggregation, as discussed below in the Examples. The enzymaticdegradation of proteins or polypeptides can be further minimized byco-incorporating peptidase-inhibitors.

[0111] Treatment of an Animal Using Slow Release Protein Polymers

[0112] The polymer articles of the present invention may be used totreat an animal, for example, a mouse, rat, or human, by delivering aBAS to the animal. The articles may contain such a BAS as any of thosedescribed above. Various routes of administration may be used to deliverthe articles of the present invention, as described below.

[0113] The results of the treatment of an animal with therapeuticarticles containing a BAS, as described herein, will vary according tothe BAS being delivered. For example, if hGH is delivered through thetherapeutic articles of the present invention, one would expect toobserve an increase in growth as a result of such a treatment. Iferythropoietin is delivered through the therapeutic articles, one wouldexpect to observe an increase in reticulocytes in the animal as a resultof the treatment. If insulin is delivered through the therapeuticarticles, then the treatment should result in a decrease in bloodglucose levels.

[0114] The articles of the present invention provide optimal delivery ofa BAS, because they release the BAS in a controlled manner with a lowburst effect. The result of such a delivery rate is that the drug isdelivered steadily over a desired period of time. A slower and steadierrate of delivery may in turn result in a reduction in the frequency withwhich the BAS must be administered to the animal. In addition, a lowburst effect may be highly desirable in some circumstances where thedelivery of too much BAS to a site is deleterious to the animal.

[0115] Routes of Administration of the Articles Inhalation

[0116] The use of the hydrogel particles of the invention can enhancethe delivery of drugs to the lung. Administration to the lung providesfor the delivery of drugs that can be transported across the lung tissuebarriers and into circulation, as described WO 99/03454.

[0117] A problem with the delivery of active substances to the lung isthat pulmonary macrophages can take up the materials, thus preventingthe material from entering into systemic and local circulation. Uptakeoccurs when proteins adsorbed to the particles' surfaces bind withreceptors on the surfaces of the macrophages. To prevent uptake, theinvention provides nonionic hydrogels, e.g., formed with polymers basedon polyethylene glycol. These hydrogels adsorb low levels of proteinsand thus bind poorly to cell surfaces. Anionic hydrogels, e.g., formedwith polyacrylic acid, also adsorb relatively low levels of proteins andthus bind poorly to cell surfaces.

[0118] In a further embodiment, biocompatible microcapsules may beformed and the surface provided with water soluble non-ionic polymerssuch as polyethylene oxide (PEO), to create resistance to cell adhesion,as described in U.S. Pat. No. 5,380,536, hereby incorporated byreference.

[0119] The size and density of the particles can also be selected tomaximize the quantity of BAS that is delivered to the lung. For example,the macrophages will not take up large particles as efficiently as theywill take up small particles. However, large particles are not deliveredto the deep lung as well as small particles are. To overcome theseconflicting factors, the invention provides small particles that canswell as they hydrate. The particles are administered to the deep lungas small (i.e., 1-5 microns), dry, or slightly wet, particles; uponhydration, they swell, and therefore become resistant to uptake by thepulmonary macrophages. The swelling can occur when the particles arehydrated from the dry state and when they are hydrated from one state ofhydration to another by a change in temperature, pH, salt concentration,or the presence of other solvents, for example, depending upon thechemical and physical nature of the hydrogel polymer.

[0120] As used herein, the term “dry” means that the particles of thepowder have a moisture content such that the powder is readilydispersible in an inhalation device to form an aerosol. Preferably, themoisture content of the particles is below 10% by weight water, morepreferably below about 5%, or optionally below about 2%, or lower.

[0121] The density of the particles is expressed in terms of tapdensity. Tap density is a standard measure of the envelope mass density.The envelope mass density of an isotropic particle is defined as themass of the particle divided by the minimum sphere envelope volumewithin which it can be enclosed. The density of particles can bemeasured using a GeoPyc (Micrometers Instrument Corp., Norcross, Ga.) ora AutoTap (Quantachrome Corp., Boyton Beach, Fla.).

[0122] For example, the density of 3.4kL5 particles was determined asfollows. 3.4kL5 (1.0025 g), 200 mM TEOA in PBS; pH 7 (1.0260 g), and1000 ppm Eosin (0.1028 g) were combined. 200 mg of this solution wasmixed with talc (0.1015 g). The resulting suspension was placed in a 100μl glass pipet and polymerized by light for 15 seconds (ILC Technology,Inc. Xenon Light Source with Fiber Optics). The rod was pushed out,placed on aluminum foil, and further polymerized for 3.5 minutes. Thehardened rod was lyophilized (vacuum 15E-3 mbar, trap temp. −50° C.) for18 hours. The dry rod (water content<10%) was cut into small pieces,placed in heptane, and minced using a homogenizer (Silverson L4RT-A) at5,000 rpm to small particles. The wet particles were air-dried, followedby nitrogen gas flow. The particles sizes ranged from 1 micron to 0.5mm.

[0123] 1.645 g of these particles was placed in a 10 mL graduatedcylinder. The graduated cylinder was mounted on top of an Autotapdensimeter (Quantachrome). The sample was tapped 100 times and theparticles' volume was read. The process was repeated until no change involume was observed. The final volume was 2.8 ml. The tap density of theparticles was 1.6435 g/2.8 ml=0.5870 g/ml.

[0124] In addition to particles, the polymer may be provided in othershapes suitable for delivery to the deep lung. For example, PEG emulsionmicrospheres are subjected to high pressure and a vacuum onto a flatplate to form very light very thin layers, for example, having a snowflake consistency, that react differently to fluidic wind forces. Theresulting thin flakes can be, e.g., 0.01 micron, 1 micron, or 10 micronsthick.

[0125] The particles can be administered to the respiratory systemalone, or in any appropriate pharmaceutically acceptable excipient, suchas a liquid, for example, saline, or a powder. Aerosol dosages,formulations and delivery systems may be selected for a particulartherapeutic application, as described, for example, in Gonda (“Aerosolsfor delivery of therapeutic and diagnostic agents to the respiratorytract,” in Critical Reviews in Therapeutic Drug Carrier Systems,6:273-313, 1990); and in Moren (“Aerosol dosage forms and formulations,”in: Aerosols in Medicine. Principles, Diagnosis and Therapy, Moren, etal., Eds., Elsevier, Amsterdam, 1985).

[0126] Pulmonary drug delivery may be achieved using devices such asliquid nebulizers, aerosol-based metered dose inhalers, and dry powderdispersion devices. For the use of dry powder dispersion devices, thepolymer particle incorporating the therapeutic agent is formulated as adry powder, for example, by lyophilization or spray-drying. Methods forpreparing spray-dried, pharmaceutical-based dry powders including apharmaceutically acceptable amount of a therapeutic agent and a carrierare described in PCT WO 96/32149, hereby incorporated by reference.

[0127] Examples of a BAS that can be administered to the lung include,without limitation, insulin, antitrypsin, calcitonin, alpha interferon,beta interferon, GLP-1, and DNAse.

[0128] Nasal Delivery

[0129] The articles of the present invention can also be used toadminister compounds nasally. For example, a vaccine containing freezedried or reconstituted microspheres can be administered nasally.

[0130] Intramuscular and Subcutaneous Administration

[0131] The articles of the present invention can be used to administermicrospheres that degrade over several days to 3 months, byintramuscular injection or by subcutaneous injection.

[0132] For example, growth hormone can be administered subcutaneously;the hormone leaves the microspheres at the site of injection as theydegrade. Growth hormone enters the systemic circulation, where, in turn,it exerts its effects directly, and indirectly through induction ofsomatomedin production in the liver and in other tissues. For thisapplication, particle sizes of up to 0.5 mm can be used.

[0133] In other embodiments, the active agent is a vaccine, such astetanus vaccine, other proteins or polypeptides, or more compleximmunogens. The vaccine is released over time, from one week to manyweeks, resulting in an improved immune response to the vaccine, comparedto a bolus injection followed by one or more booster shots with the sametotal dose of immunogen. Mixtures of different types of microspheres canresult in initial and booster shot-type immunization as well.

[0134] Intravenous Administration

[0135] Articles that contain a BAS useful in treating clottingdisorders, such as Factor VIII or Factor IX for hemophilia, can beadministered by intravenous injection. The BAS is released over days toweeks. A therapeutic level of the BAS is maintained that results in abetter clinical outcome. In addition, potentially lower total doses of aBAS can be administered, with a corresponding economic benefit. Theseapproaches help promote patient compliance.

[0136] In the case of intravenous injection, it is important toformulate the microspheres in acceptable agents so the microspheres donot aggregate and clog blood vessels. The microspheres must beappropriately sized, so that they don't lodge in capillaries. For thisapplication, particle sizes of 0.2-0.5 microns are preferred.

[0137] In a number of inflammatory conditions, as part of theinflammatory process that is mediated by selectin and ICAMexpression/binding with neutrophil intravisation, blood vessels becomeleaky at the site of inflammation. Hydrogel microspheres may beadministered; these microspheres will leak out of blood vessels at thesite of inflammation, and then release their BAS payload locally over aperiod of time. Disease conditions where this approach may be usefulcould include, but are not limited to, inflammatory bowel diseases,asthma, rheumatoid arthritis, osteoarthritis, emphysema, and cysticfibrosis (with DNase as the enzymatic drug).

[0138] Hydrogel microspheres that contain cytokines, lymphokines, orother compounds to treat cancer can be administered by intravenousinjection. Blood vessels within large solid tumors are generally leaky,and the blood flow within them is often slow. Thus, microspheres couldlodge within solid tumors and release their anticancer BAS locally,either killing tumor cells directly or by activating the immune systemlocally. This approach could be used, for example, with compounds suchas interleukin 2, where the systemic and local toxicity has been doselimiting and where the resulting side effects are significant.

[0139] The microspheres of the present invention may be clearedrelatively slowly from the circulation. Alternatively, the microspherescan be targeted to exit the circulatory system through leaky bloodvessels or through more active targeting mechanisms, e.g., receptormediated targeting mechanisms.

[0140] Oral Administration

[0141] In some portions of the gastrointestinal tract, there isrelatively good transport of proteins across the intestinal mucosa intothe systemic and local circulation. The compositions of the invention,for example, freeze dried microspheres containing protein (with verysmall particle sizes), can therefore be administered orally in anappropriate enteric formulation that protects the drug-containingmicrospheres from enzymatic attack and the low pH found in the upper GItract. Such an enteric formulation could also be designed using severalavailable technologies to gradually expel BAS-containing microspheres asthe enteric capsule traverses the gastrointestinal tract. This isdescribed in more detail in WO 99/03454 and in Mathiowitz et al. (Nature386: 410-414 (1997)). It is anticipated that this approach will have anumber of advantages over other approaches for delivering proteins andother molecules, even small molecules, orally. First, PEG and proteinsare compatible, so the major manufacturing and stability problems foundwith other drug delivery approaches can be avoided. Secondly, driedhydrogels are very adhesive to wet tissue. The microparticles will bindwell to the GI tract and will be transported into the system via thegastrointestinal circulation or release their contents on the intestinalmucosa; in turn, the drug will enter the systemic and gastrointestinalcirculation. Chemical enhancers, or formulations containing compositionsthat utilize specific and non-specific biological transport mechanismsto facilitate transport across the GI tract into the systemiccirculation, can be included as well.

[0142] Targeting

[0143] Targeting ligands can be attached to the particles via reactivefunctional groups on the particles. Targeting ligands permit bindinginteractions of the particle with specific receptor sites, such as thosewithin the lungs or those on endothelial cells specific to differentregions in the body's microvasculature. A targeting ligand is selectedwhich specifically or non-specifically binds to particular targets.Exemplary targeting ligands include antibodies and fragments thereofincluding antibody variable regions, lectins, hormones, or other organicmolecules capable of specific binding to receptors on the surfaces ofthe target cells. Other ligands are described in Science (279:323-324(1998)), hereby incorporated by reference.

[0144] Microspheres can be made with both a BAS and a targetingmolecule. Double microspheres can also be made, in which the innersphere contains drug and the outer PEG shell contains the targetingmolecule or reagent.

[0145] Excipients and Carriers

[0146] The particles incorporating a therapeutic agent or diagnosticagent may be provided in combination with one or more pharmaceuticallyacceptable excipients available in the art, as described, for example,in PCT WO 95/31479, hereby incorporated by reference. Excipients may beselected that can, in some applications, enhance stability,dispersability, consistency, and bulking to ensure uniform pulmonarydelivery. The excipient may be, e.g., human serum albumin (HSA), bulkingagents such as carbohydrates, amino acids, polypeptides, pH adjusters orbuffers, and salts. Additional excipients include zinc, ascorbic acid,mannitol, sucrose, trehalose, cyclodextrans, polyethylene glycol, andother commonly used pharmaceutical excipients, including those describedin The United States Pharmacopeia, published by the United StatesPharmacopeia Convention, Inc., 1995 (see, e.g., pp. 2205-2207).Exemplary carbohydrates include monosaccharides, such as galactose, anddisaccharides such as lactose. Excipients that stabilize proteins areespecially useful.

[0147] In some cases, the excipients are used as carriers; i.e., theyare used to modulate the release rate of the active substances. Forexample, mannitol can be used to accelerate or delay release.

[0148] There now follow particular examples that describe thepreparation of compositions of the invention, and the methods of theinvention. These examples are provided for the purpose of illustratingthe invention, and should not be construed as limiting.

[0149] In some of the following Examples a macromer made of a triad ABAblock copolymer of acrylate-PLA-PEG-PLA-acrylate was used. The PEG had aMW of 3,400 daltons; the poly(lactic acids) on both sides had an averageof about five lactate units per side; they are therefore referred toherein as “3.4kL5.” When a lower molecular weight PEG, such as 2,000daltons was used, the resulting macromer is abbreviated as “2kL5.”

[0150] In other Examples an acrylate-PCL-PEG-PCL-acrylate macromer wasused. The PEG had a MW of 3,400 daltons and had polycaprolactone on bothsides, with an average of about 6 caproyl units per side. The polymer isreferred to herein as “3.4kC6.”

[0151] In yet other Examples a 3-arm macromer was used. This macromerconsisted of a PEG core with 3 arms, 3 lactate groups attached to eacharm of the PEG. The PEG had a MW of 4,200 daltons and the polymer isreferred to herein as “4.2kL3-A3.”

EXAMPLE 1

[0152] General Preparation of a Macromer Solution

[0153] The protein was weighed out, and the following components wereadded to the protein: (i) 90 mM TEOA/PBS, pH 8.0; (ii) 35% n-vinylpyrrolidinone (n-VP); and (iii) 1000 ppm Eosin. The resulting mixturewas stirred well using a spatula. The solution was kept in the dark forabout 10 minutes, or until the macromer had absorbed all of thesolution, or until the solution was homogenous.

[0154] Macromer solutions having the following ingredients wereprepared. Amount Amount Amount Amount 90 mM 35% 1000 ppm Amount AmountTotal Protein TEOA n-VP Eosin 3.4kL5 2kL5 amount 15 mg 57 mg 15 mg 3 mg45 mg  0 mg 135 mg 15 mg 57 mg 15 mg 3 mg  0 mg 45 mg 135 mg

EXAMPLE 2

[0155] Precipitation of hGH and Formulating into Hydrogel Microspheres

[0156] To a 100 mg/ml hGH solution in 5 mM ammonium hydrogen carbonatebuffer 77 μl of a 1300 mM triethanolamine, pH 8, solution was added.Upon the addition of 400 mg of PEG 2K to the above solution, a fineprecipitate of hGH was formed. The sample was centrifuged at 4000 rpmfor several minutes and 0.9 ml of the supernatant was removed. To theprecipitated mixture, 1 g of 4.2kL5-A3 macromer was added, followed bythe addition of 0.1 ml of a 10 mM Eosin Y solution. The mixture was thenemulsified in an oil phase to form microspheres which were polymerizedusing an argon laser. The in vitro release characteristics of thisformulation are shown in FIG. 1. No burst was observed and releasecontinued for at least 5 days.

EXAMPLE 3

[0157] Micronization of Freeze-Dried Human Growth Hormone andFormulation into Hydrogel Microspheres

[0158] A 10 mg/ml solution of hGH ammonium acetate was frozen andfreeze-dried, resulting in a dry cake of pure hGH. The followingmacromer solution was prepared: 1 g 2kL5, 1.2 g phosphate bufferedsaline (pH 7), 0.4 g of a solution of 25% trehalose and 0.4% F-68 inwater, 0.24 g of a 10% solution of 2,2-dimethoxy 2 phenyl-acetophenonein tetrahydrofuran. To this solution was added 0.2 g of the freeze-driedhGH. Following mixing to disperse the hGH, the hGH in suspension wasfurther micronized by 20 passages through a 1.5 inch 20 g needle. Thissuspension was then emulsified in an oil phase to form microsphereswhich were polymerized by exposure to UV light (365 nm) for 3 min. Thein vitro release characteristics of this micropheres are shown in FIG.2.

EXAMPLE 4

[0159] Formation of Articles

[0160] The microspheres, in the form of a hydrogel, were placed onto asilanized glass slide. Using pieces of plastic sheets with thicknessesof about 0.4±0.2 mm as spacers, another silanized glass slide was placedon top and held firmly in place using binder clips.

[0161] A light source (ILC Technology, Inc. Xenon Light Source withFiber Optics) was adjusted to about a 5-cm distance. The center of thedisk was illuminated; both sides of the disk were illuminated for twominutes each, to form an opaque disk.

EXAMPLE 5

[0162] Preparation of Articles Containing 4.2kL3-A3 Macromers and bSA

[0163] To 1 ml of 50% ethanol/water solvent, 1 g of 4.2kL3-A3 macromerwas added with 7.8 mg of 2,2-dimethoxy-2-phenyl-acetophenone (DMPA). Oncomplete dissolution of the macromer and DMPA, 250 mg of spray dried bSAwas added and stirred until the mixture was uniform. The entire mixturewas then emulsified in 200 g of a 0.5% lecithin in heavy white mineraloil solution stirred at 600 rpm. Shortly afterwards, the disperseddroplets were photo-polymerized using a Black-Ray B100AP UV lamp for aperiod of 15 minutes. The in vitro release profile of this formulation,shown in FIG. 3. indicates no burst.

[0164] A degradable macromer (4.2kL3-A3) was combined with bSA. Theprotein was loaded at a loading of 20%, based on dry weight. An emulsionwas formed using white heavy mineral oil. Polymerization of the macromerinto a hydrogel then occurred through spray drying and UV polymerizationtechniques.

EXAMPLE 6

[0165] Analyses of Biological Active Substance Release from a Macromer

[0166] After formation of the articles, as described, for example, inExample 4, the disks were removed and weighed on a clean, taredsilanized glass slide. The disk was placed into a heat-sealed membranebag, as described in more detail below. One 20 μl disk was placed ineach bag. The bag was heat-sealed, placed in 2.0 ml of phosphate bufferrelease media (0.01% NaN₃, 0.05 M PBS; pH 7.4), placed on an orbitalshaker turning at 100 rpm, and incubated at 39° C.

[0167] For each time point, the bag was placed into fresh 2.0 ml of PBSRelease Media. Samples were collected for analysis every day for as longas the BAS was being released.

[0168] Membrane bags were prepared as follows. Membrane sheets were cutinto pieces of approximately 7×2.5 cm. The sheets were folded in half.Using a Bunsen burner or a propane torch, a spatula was heated until itbecame red. The edges of the sheets were aligned, and the side of themembrane was cut with the red-hot tweezer to seal the sides. Once thedisk was placed into the bag, the last side was sealed using the sameheat-sealing technique.

[0169] The samples were analyzed daily by SEC-HPLC. Monomers, dimers,and soluble aggregates could be detected using this method. The mobilephase used was 0.08 M TFA in 60/40% CH₃CN/H₂O, adjusted to pH 2.0,isocratic, with a flow rate of 1.5 ml/min. The signals were detection ata wavelength of 220 nm. The column used was a Bio-Rad Bio-Sil® SEC 250,5 micron particle size, 300×7.8 mm ID, equipped with a guard column(Bio-Rad Bio-Sil® R SEC 250 Guard, 5 micron particle size, 80×7.8 mmID). The injection volume was 10 μl. The standard calibration curveswere 0, 0.1, 0.25, 0.5, 0.75, and 1 mg/ml bST in the mobile phase.

EXAMPLE 7

[0170] Production of Microspheres with Efficient Protein Loading and LowBurst Effects

[0171]FIG. 3 shows an example of the high protein loading and low burstcharacteristics of the therapeutic articles of the present invention.The articles contain 4.2kL3-A3 macromers combined with bSA (in either amonomer or dimer form) and formed into microspheres using spray dryingtechniques. The bSA was loaded at a calculated loading of 20%. Releaseof bSA from the microspheres was assayed as described above. The releaseoccurred at a slow steady rate, and no burst effect was exhibited. Aftera period of 9 days, less than 30% of the bSA was released from macromerscontaining bSA. These results demonstrate that the therapeutic articlesof the present invention can provide slow release of a BAS, with littleor no burst effect.

EXAMPLE 8

[0172] Effect of the Particle Size of a BAS on Release of bSA from 30%3.4kL5

[0173] The effect of the particle size of a BAS on its release from anarticle, for example, a microsphere was also determined. The bSA used toform the microspheres was either ground under liquid nitrogen into fineparticles of less than approximately 75 microns, or were left unground.Microspheres containing 30% 3.4kL5 and either the fine-ground orunground bSA were formed using the methods described above, and wereassayed for the release of bSA, loaded at 25%, based on dry weight. FIG.4 illustrates the results of these studies. Compared to the microspherescontaining unground bSA, the microspheres containing fine-ground bSAreleased its bSA over a longer period of time. In addition, themicrospheres containing the fine-ground bSA exhibited steady rate ofrelease (releasing less than 20% of the total bSA loaded within thefirst 24 hours), with no burst effect, while the microspheres containingthe unground bSA exhibited a burst effect (releasing approximately 50%of the total bSA loaded within 24 hours). These results demonstrate thatmicrospheres containing a BAS which has a small particle size provideslower release profiles and low burst effects.

EXAMPLE 9

[0174] Effects of BAS Particle Size and Protein Loading on the Releaseof Fine-Ground bSA (10% Loaded) and Various Crystalline Particles (1-10%Loaded) from 3.4kL5

[0175] The effects of the particle size of a BAS and protein loading onthe release of bSA from an article, was also examined (FIG. 5). The bSAused to form the microspheres was either ground under liquid nitrogeninto fine particles of less than approximately 75 microns, or were leftunground. The fine-ground bSA was combined with 3.4kL5 to form 30%3.4kL5 microspheres loaded with 10% bSA, using the methods describedabove. The unground bSA, containing various particle sizes was combinedwith 3.4kL5 to form 30% 3.4kL5 microspheres loaded with 1-10% bSA. Themicrospheres were then assayed for the release of bSA. FIG. 5illustrates the results of these studies. Compared to the microspherescontaining unground bSA, the microspheres containing fine-ground bSA,loaded at 10%, released its bSA over a longer period of time. Inaddition, the microspheres containing the fine-ground bSA exhibited alower burst effect (releasing approximately 20% of the total bSA loadedwithin the first 24 hours) than its unground counterpart (releasingalmost 50% of the total bSA loaded within the first 24 hours). Theseresults demonstrate that microspheres containing a BAS which has a smallparticle size and is highly loaded provide a desirable BAS releaseprofile compared to microspheres containing various particle sizes and alower load of BAS.

EXAMPLE 10

[0176] Effect of Protein Particle Size on Release of hGH from 30% 3.4kL5

[0177] The effect of the particle size of a BAS on its release frommicrospheres was further examined using microspheres containing eitherfine-ground or unground hGH. The hGH used to form the microspheres waseither ground under liquid nitrogen into fine particles of less thanapproximately 75 microns, or were left unground. Microspheres containing30% 3.4kL5 and either the fine-ground or unground hGH were formed usingthe methods described above, and were assayed for the release of hGH,loaded at 25%, based on dry weight, and 10% as manufacturing conditions.FIG. 6 illustrates the results of these studies. Compared to themicrospheres containing unground hGH, the microspheres containingfine-ground hGH released its hGH over a longer period of time. Inaddition, the microspheres containing the fine-ground hGH exhibited asteady rate of release (releasing less than 40% of the total hGH loadedwithin the first 24 hours), with no burst effect, while the microspherescontaining the unground hGH exhibited a high burst effect (releasingapproximately 70% of the total hGH loaded within 24 hours). Theseresults demonstrate that microspheres containing a BAS which has a smallparticle size provide the characteristics which are highly suitable fordelivery of agents for therapeutic use: slow protein release and lowburst effects.

EXAMPLE 11

[0178] Effect of Microsphere Pore Size on Release of hGH from Macromers

[0179] The effect of the pore size of the microspheres containingfine-ground hGH on the release rate of the hGH was also examined (FIG.7). Microspheres containing hGH, loaded at 25%, based on dry weight, and10% as manufacturing conditions, and either 30% 2kL5 or 30% 3.4kL5 wereformed using the techniques described above. The microspheres containing2kL5 had a smaller pore size than the microspheres containing 3.4kL5.The release rate of hGH from these microspheres was then assessed usingtechniques described above. These studies showed that fine-ground hGHwas release from 2kL5-containing microspheres at a slower rate than itwas released from 3.4kL5-containing microspheres. These results suggestthat macromers which result in a smaller microsphere pore size release aBAS at a slower rate than those which result in a larger microspherepore size.

EXAMPLE 12

[0180] Controlled Release of Bovine Somatotropin in HypophysectomizedRats

[0181] The controlled delivery of active bovine somatotropin (MW 20 Kd)was confirmed in the hypophysectomized rat model. Hypophysectomizedfemale rats were purchased from Taconic Labs (Germantown, N.Y.). Therats were weighed each morning. Prior to the initiation of the study,the rats were held 7 days to confirm a lack of significant growth. Onday 1 of the study the rats were weighed. The rats were then dividedinto 3 groups of equal mean weights. Group 1 remained untreated andserved as a negative control. Group 2 received an implant of bST in ahydrogel made of a blend of 3:1 of 3.4KL5 and poly(ethylene glycol)diacrylate (each device contained 0.9 to 1.1 mg of bST). The rats inGroup 3 were injected with 100 μg bST subcutaneously each day for theduration of the study.

[0182] At the end of the 12 day treatment period, the rats were analyzedfor their growth over the period of treatment. The rats of Groups 1 didnot grow significantly, while the rats of Groups 2 and 3 grew at ratesfaster than Group 1 and approximately equal to one another.

EXAMPLE 13

[0183] Controlled Release of Erythropoietin in Rats

[0184] The controlled delivery of active human erythropoietin (EPO) wasconfirmed in male Sprague-Dawley rats purchased from Taconic Labs(Germantown, N.Y.). Hydrogel devices were manufactured to contain 3000Units of EPO per device. One of these devices was implanted in each of anumber of rats (Group 1). Another group of an equal number of rats(Group 2) received a subcutaneous injection of EPO (1000 Units) dailyfor 3 days. A third group of rats (Group 3) received no treatment.

[0185] On day 5 after implantation of the device and the start of thesubcutaneous injections, venous blood samples were obtained from eachrat and stored in EDTA. The fraction of reticulocytes (immature redblood cells) was determined after staining with Acridine Orange byautomated flow cytometry. The rats in Group 1 had 18% reticulocytes, therats of Group 2 had 15% reticulocytes, and the rats in Group 3 had 4%reticulocytes.

EXAMPLE 14

[0186] Controlled Release of Insulin in Diabetic Rats

[0187] Sprague-Dawley rats were purchased from Taconic Labs (Germantown,N.Y.). Diabetes was induced by treatment with streptozotocin (65 mg/kg,i.v.) and confirmed 48 hours later by elevation of blood glucose (>300mg/dl). Following anesthetization of the rat with pentobarbital (35mg/kg), a catheter was placed in a jugular vein. After a baseline bloodsample was taken for the determination of blood glucose concentration, ahydrogel device containing 1 Unit of insulin was implantedsubcutaneously. Blood samples were taken at 15, 30, 60, 120, and 180minutes after implantation of the device and used to determine bloodglucose levels.

[0188] The blood glucose level of the rat implanted with the hydrogeldevice decreased, demonstrating that the devices was capable ofreleasing insulin in its active form.

[0189] To test the pulmonary delivery system for insulin-containinghydrogel particles, the neck of the rat was opened with a midlineincision and the trachea was exposed by blunt dissection. A slit was cutinto the trachea, and a small polyethylene tube was advanced distallyinto the lung. A small volume of insulin-containing hydrogelmicroparticles (total dose of 3 Units of insulin) was instilled into thelung and the tube was removed. Blood samples were taken and analyzed asdescribed above for the subcutaneous device.

[0190] The blood glucose levels dropped significantly within 30 minutesand remained low (below 150 mg/dl) for at least 180 minutes.

EXAMPLE 15

[0191] Controlled Release of Human Growth Hormone in HypophysectomizedRats

[0192] The controlled delivery of active human growth hormone (hGH, MW20 Kd) is confirmed in the hypophysectomized rat model.Hypophysectomized female rats purchased from Taconic Labs (Germantown,N.Y.) are weighed each morning. Prior to the initiation of the study therats are held 7 days to confirm lack of growth. The rats are dividedinto 3 groups of equal mean weights. Group 1 remains untreated andserves as a negative control. Group 2 receives an implant of hGH in ahydrogel made of a 3:1 blend of 3.4kL5 and 3.4kC6 (each devicecontaining approximately 1 mg of hGH). The rats in Group 3 are injectedwith 100 μg hGH subcutaneously each day for the duration of the study.

[0193] It is expected that the untreated control group will not growduring the study, and that the rats of Groups 2 and 3, receiving the hGHhydrogel implant and 100 μg hGH injections daily during the study,respectively, will exhibit continued growth.

EXAMPLE 16

[0194] Pulmonary Devices Containing Human Growth Hormone (hGH)

[0195] To a 20 ml vial are added: 0.2559 g of 200 mM of TEOA (in PBSbuffer; pH 7.0), 0.2548 g of 3.4KL5, 0.0206 g of 1000 PPM eosin (in PBS;pH 7.0), and 0.0615 g hGH (Genentech's hGH injectable formulation,purified by a Millipore Centricon™). The resulting mixture is stirredand placed into 10 ml glass tubes. The tubes are exposed to xenon light(ILC Technology, Inc. Xenon Light Source with Fiber Optics) for 10seconds. The semi-cured hydrogel is pushed out of the glass tube andfurther polymerized for 3.5 minutes. The cured hydrogel rods are putinto 15 ml of heptane and are ground using a homogenizer (SilversonL4RT-A) for 30 seconds at 5000 rpm, followed by 30 seconds at 3000 rpm.The heptane is decanted, and the powder is dried under nitrogen. Thepowder is used for pulmonary, oral, or subcutaneous sustained deliveryof hGH.

EXAMPLE 17

[0196] Oral Formulation for Release of Proteins

[0197] Using the techniques described above, insulin, human growthhormone, human alpha interferon, or erythropoietin is incorporated intomacromer particles. Using cryomilling or other milling procedures knownin the art, very small microparticles are produced, preferably of anaverage size of less than about 500 nanometers. Such nanoparticles arethen introduced into the rat GI tract surgically, using catheterinfusion into the upper GI tract. The dosing of such nanoparticles isbased upon the assumption that about 0.5% of the drug in thenanoparticles will be detectable in the blood of such rats, e.g., byRIA, with the specific pharmacology of each drug taken into account.

[0198] In the case of insulin, blood samples are taken at time t=−15, 0,30, 60, 90, 120, and 180 minutes, and monitored for insulin by RIA andfor blood glucose by glucometer (when insulin is being administered,diabetic rats are utilized).

[0199] For other drugs, normal rats are used and blood drug levels aremeasured at these same time points using RIA or ELISA techniques.

[0200] In addition to the above procedures, the above drug-containingmicrospheres can be modified to enhance their absorption in the smallintestine, colon, and other appropriate areas of the GI tract. Suchmodifications can include precipitating lipid bilayers around themicrocapsules so they appear as fat-like particles from digested food,linking molecules such as ferritin to the particles, or putting acharged layer on the outside of the microparticles.

EXAMPLE 18

[0201] Evaluation by Reverse Phase HPLC

[0202] Microspheres were prepared by first adding 0.154 ml of 3Mtriethanolamine solution at pH 8.0 (TEOA) to approximately 2 ml of 100mg/ml solution of hGH in ammonium bicarbonate and then mixing well.Next, about 800 mg of solid PEG 2k was added and mixed with a spatula,resulting in a very small amount of precipitated hGH in the solution.Samples were then centrifuged at 4000 rpm for thirty minutes and about1.8 g of supernatant was removed. About 1 g of macromer (4.4k PEGtris(lactate)₃ triacrylate) was added to each centrifuge tube andstirred. Next, about 0.1 to 0.15 mL of TEOA was added, followed by 0.05mL of 40 mM Eosin Y. The samples were then centrifuged for three minutesat 4000 rpm. Samples were then polymerized by forming a disc uponexposure to light as described in Example 4 or by first making amicroemulsion by mixing with oil (PPG 2k) and then exposing to light asdescribed in Example 4.

[0203] The resulting microspheres were analyzed by Reverse Phase HPLC(RP-HPLC). Samples were prepared for RP-HPLC by first extracting hGHfrom the microspheres with NaOH. Briefly, 10 mg of microspheres wasadded to 1 mL of NaOH(1N)/Tris (50 mM, pH 7.5) (1:9, v/v) solution andincubated at ambient temperature for 5 minutes. 5N HCl was used totitrate the solution to a final pH of 7.5. The sample was thenmicrocentrifuged for 2 minutes and filtered through a 0.45 micronfilter. 100 μL of the hGH solution was injected onto a Vydac C-4 column(214TP54) equilibrated and run under isocratic conditions at 0.5 mL/min.employing n-propanol/Tris (50 mM, pH 7.5)(29:71, v/v) as a solvent.Separation was performed at column temperature of 45° C. over 50 minuteswith UV detection at 220 nm. hGH eluted at a retention time of 33±3minutes. Results are shown in Table I. The term “% RP” refers to thepercentage of protein (hGH) that is not found in the monomer peak, whichmay include forms of hGH that are normally found in commerciallymarketed hGH preparations and that are active and safe, such as oxidizedand deamidated forms. TABLE I Sample No. % RP  18-86-1 41 318-1 48 318-243

[0204] Other batches of microspheres were prepared by first adding 0.154ml of 3M Tris buffer at pH 6.0 to about 2 ml of 100 mg/ml hGH solutionin ammonium bicarbonate and mixing well. To this solution, about 800 mgof PEG 10k was added as solid to obtain precipitated protein. Sampleswere then centrifuged at 4000 rpm for thirty minutes and about 1.8 g ofsupernatant was removed. About 1 g of macromer (4.4k PEG tris(lactate)₃triacrylate) was added to each centrifuge tube and stirred. Next, about0.1 to 0.15 mL of TEOA was added, followed by 0.05 mL of 40 mM Eosin Y.The samples were then centrifuged for three minutes at 4000 rpm. Sampleswere then polymerized by forming a disc upon exposure to light asdescribed in Example 4 or by first making a microemulsion by mixing withoil (PPG 2k) and then exposing to light as described in Example 4. Onesample, Sample No. 27-50, was prepared using PEG 20k.

[0205] The resulting microspheres were analyzed by Reverse Phase HPLC.Samples were prepared for RP-HPLC either by the NaOH extraction methoddescribed above or by the following cryogrinding method. Results areshown in Table II, with the sample preparation method indicated.Approximately 10 mg of microsphere sample was weighed in amicrocentrifuge tube. A pestle was placed in the tube and then the tubewas immersed in a liquid nitrogen bath for approximately one minute.With the tube still in the liquid nitrogen bath, the sample was groundfor approximately five minutes. The tube was then removed and allowed tostand for approximately two minutes at ambient temperature. The groundsample was then suspended in about 1 mL of 25 mM potassium phosphatebuffer, pH 6.5. The pestle was removed, and the sample incubated forabout ten minutes at ambient temperature. The sample was thencentrifuged at 15000 rpm for about five minutes to obtain a clearaqueous phase. The supernatant was the filtered through a 0.45 micronfilter. RP-HPLC was performed by injecting 100 μL of sample onto a VydacC-18 column (218TP54) equilibrated and run under isocratic conditions at1 mL/min, using n-propanol/potassium phosphate (25 mM, pH 6.5) (27:73,v/v) as a solvent. Separation was performed at a column temperature of55° C. over 30 minutes with UV detection at 220 nm. TABLE II Sample No.% RP 27-32 26 (NaOH) 27-50 19 (NaOH) (Tris, PEG 20k) 320-4  20 (NaOH)502 15.2 (NaOH) 507 17 (NaOH) 508 15 (NaOH) 530 16 (NaOH) 548 10.4(Cryogrind) 556 7.7 (Cryogrind) 557 8.7 (Cryogrind) 561 8.5 (Cryogrind)570 6.2 (Cryogrind) 575 7.2 (Cryogrind)

[0206] By comparison, microspheres made without a molecule thatpreferentially excludes proteins (“Control” in Table III), yield % RPvalues of 53% using the NaOH extraction method, and 23% using thecryogrind method. Also presented in Table III is a comparison of the twosample preparation methods. TABLE III Sample No. % RP NaOH Method % RPCryogrind Method Control 53 23 361 30.2 15 362 28 15

Other Embodiments

[0207] From the foregoing description, it will be apparent thatvariations and modifications may be made to the invention describedherein to adopt it to various usages and conditions. Such embodimentsare also within the scope of the following claims.

[0208] All publications and patents mentioned in this specification areherein incorporated by reference to the same extent as if eachindividual publication or patent was specifically and individuallyindicated to be incorporated by reference.

What is claimed is:
 1. A biocompatible therapeutic article comprising, amacromer, a polypeptide, and a molecule or mixture of molecules whichpreferentially excludes proteins, wherein said polypeptide is insolublein said article, wherein said polypeptide is selected fromparathyroid-related polypeptide, somatostatin, luteinizing hormonereleasing hormone, GLP-1 amylin and GLP-1 amyline analogues.
 2. Thebiocompatible therapeutic article of claim 1, wherein said moleculewhich preferentially excludes proteins is selected from the groupconsisting of a macromer, poly(ethylene glycol), hyaluronic acid, andpoly(vinylpyrrolidone).
 3. The biocompatible therapeutic article ofclaim 1, wherein said macromer comprises: (a) a region forming a centralcore; (b) at least two degradable regions attached to said core; and (c)at least two polymerizable end groups, wherein said polymerizable endgroups are attached to said degradable regions.
 4. The biocompatibletherapeutic article of claim 3, wherein said central core comprises apolymer selected from the group consisting of poly(ethylene glycol),poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone),poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide)block copolymers, polysaccharides, carbohydrates, proteins, andcombinations thereof.
 5. The biocompatible therapeutic article of claim3, wherein said degradable regions comprise a polymer selected from thegroup consisting of poly(α-hydroxy acids), poly(lactones), poly(aminoacids), poly(anhydrides), poly(orthoesters), poly(orthocarbonates), andpoly(phosphoesters).
 6. The biocompatible therapeutic article of claim1, wherein said polypeptide is parathyroid-related polypeptide.
 7. Thebiocompatible therapeutic article of claim 1, wherein said polypeptideis somatostatin.
 8. The biocompatible therapeutic article of claim 1,wherein said polypeptide is luteinizing hormone releasing hormone. 9.The biocompatible therapeutic article of claim 1, wherein saidpolypeptide is GLP-1 amylin or GLP-1 amyline analogues.
 10. Thebiocompatible therapeutic article of claim 1, wherein said articlecomprises at least 5% polypeptide by dry weight.